Approximately 500,000 Americans have end stage renal disease, in which kidney function is insufficient to sustain life. Organ function can be supplemented by dialysis in these individuals; however the 10 year survival rate for individuals on dialysis is just over 10%. Survival rates are much better for patients receiving a kidney transplant but organ supply does not match demand. Ex vivo organogenesis has the potential both to provide functional tissue for renal replacement therapy and to provide research tools with which we can understand the causes of chronic kidney disease and identify new therapies. Furthermore, defining signals that functionally direct nephrogenesis may identify pathways that can be manipulated to augment the regenerative response of the injured kidney in vivo. Our group is able to promote nephrogenesis in cultures of purified primary nephron progenitor cells derived from human stem cells and mouse embryonic kidneys. However, we have identified two key obstacles that must be overcome if this discovery is to have significant impact on human health. First, we must recreate signaling environments that promote progenitor cell proliferation and differentiation in order to obtain sufficient tubule mass for functional analysis. Second, we must devise ex vivo tissue architecture that supports differentiation of arrayed nephrons with vascular connections that are appropriately patterned. The long-term goal of this proposal is to define the tissue architecture and cell signaling microenvironments required to promote the generation of appropriately patterned nephrons in culture. We will investigate signaling mechanisms that are sufficient to promote NPC renewal and differentiation and apply these findings to the development of ex vivo nephron devices using silk protein as a scaffolding biomaterial. Our data show that NPCs efficiently colonize silk and that nephron tubules form in this material. Silk is the material of choice because it is already in clinical use and it is scalale, allowing us to generate structures dimensioned for mouse or human. We will leverage the strengths of the project investigators in kidney and endothelial development as well as bioengineering to develop an integrated, multi-cellular, patterned, functional nephron in vitro. State of the art, innovative technologies will be applied to NPCs differentiated from both human embryonic stem cells and embryonic mouse kidneys. Novel scaffolding and matrix formulations that allow organized 3D organogenesis of tissues in culture will be explored. The investigative group has a 10-year history of collaboration, which is particularly evident in the integrated natur of our preliminary data. We will leverage this program to develop a seamless exchange of engineering and signaling expertise, materials and intellectual innovation between all four laboratories. The program therefore includes a framework for continual scientific exchange including a data tracking website, a bi-monthly schedule of group meetings and face-to-face meetings approximately every 6 months.